Mammalian blood cells provide for an extraordinarily diverse range of activities. Hematopoietic stem cells are defined as those cells that are capable of both self-renewal and differentiation into the two principle precursor components—the myeloid and lymphoid lines. Such stem cells are said to be “totipotent.” Stem cells that are less general but that can still differentiate into several lines are called “pluripotent.” Further differentiation then occurs among the precursor cells to produce the monocyte, eosinophil, neutrophil, basophil, megakaryocytes, and erythroid lineages from the myeloid line, and T cells, B cells, and NK cells from the lymphoid line. Hematopoietic stem cells reside primarily in the bone marrow.
One of the first breakthroughs into stem cell isolation and identification came in the late 1980's. In U.S. Pat. No. 4,714,680, Civin described pluripotent lympho-hematopoietic cells that were substantially free of mature lymphoid and myeloid cells. Civin also described an antigen, MY-10, and a monoclonal antibody thereto, which was present on those cells. Those cells made up about 1% of all cells in normal adult bone marrow, and generally comprised a mixture of totipotent, and pluripotent stem cells and lineage committed precursor cells with the latter cells predominating. Since that time, MY-10 has been classified by the International Workshop on Human Leukocyte Antigens as falling with the cluster designated as “CD34.” Anti-CD34 monoclonal antibodies are now commercially available from a number of sources including, for example, Becton Dickinson Immunocytometry Systems (“BDIS”).
Other investigators have attempted to subset CD34+ cells from both peripheral blood and bone marrow. Bender et al., Blood 77:2591–2596 (June 1991), used four color flow cytometry with combinations of monoclonal antibodies (i.e., anti-CD34, anti-CD33, anti-CD45, anti-CD19, anti-CD7, anti-CD10, anti-CD3, anti-CD20, anti-CD14, anti-CD11b and anti-HLA-DR), to identify and isolate CD34+ hematopoietic progenitor cells.
There is a strong interest in identifying and isolating human hematopoietic stem cells. However, the stem cell population constitutes only a small percentage of the total number of leukocytes in bone marrow. In view of the small proportion of the total number of cells in the bone marrow which are stem cells, the uncertainty of the markers associated with the stem cell as distinct from more differentiated cells, and the general inability to biologically assay for human stem cells, the identification and purification of stem cells has been difficult.
Having a procedure for the efficient isolation of stem cells would allow for identification of growth factors associated with, for example, (1) the early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the negative control of stem cell proliferation. Readily available stem cells would also be extremely useful in bone marrow transplantation, as well as transplantation of other organs in association with the transplantation of bone marrow. Also, stem cells are important targets for gene therapy, where the inserted genes promote the health of the individual into whom the stem cells are transplanted. In addition, the ability to isolate stem cells may serve in the treatment of lymphomas and leukemia, as well as other neoplastic conditions. The identification and isolation of the most primitive population of hematopoietic stem cells would be highly advantageous in situations where reinfusion of only a small number of long-term repopulating cells was desired. For example, this would be the case when purging bone marrow or peripheral blood stem cells of contaminating tumor cells, or where genetic manipulation of the stem cells was the objective.
The separation of a particular mammalian cell population from a mixture of cell populations is quite different from the separation of chemical species such as proteins from a solution. Most mammalian cells are on the order of 8 to 20 microns (μ) in diameter. In contrast, the proteins and other chemical species are significantly smaller, i.e., on the order of 1000-fold or more. In addition, another factor unique to the separation of mammalian cells is the need to preserve cell viability.
In contrast to yeast cells, which are relatively insensitive to changes in osmolarity, pH and shear, higher order mammalian cells are much more sensitive to shear forces-exerted during purification, pH osmolarity, and the chemical composition of the reagents used. Therefore, the steps comprising the method and all reagents used must be non-toxic to the cells.
Separation of mixtures of chemicals, biomolecules and cell types is often effected by immunoaffinity chromatography. Packed beds, such as those used in column chromatography, are often used in affinity separation. However, problems such as non-specific trapping or filtration of cells and clogging make the use of a packed bed undesirable for cell separation.
One device that has been developed for reducing the pressure drop across a column of particles is the fluidized bed. A fluidized bed consists of solid particles and a gas or liquid which is passed upwardly through the particle bed with velocity sufficient to fluidize the bed. The fluidization of the bed provides more surface contact between the particle and the fluid passing through the bed. One disadvantage associated with fluidized beds is the radial and axial movement of the particles which result in significant intermixing of the particles.
A number of other methods have been developed for fractionating heterogeneous mixtures of cells into the various compartments. These methods are based on the size and density of the cells, specific binding properties that they possess, and their expression of surface antigens. The method chosen usually depends on the degree of purity required, the intended use of the selected cells, and the abundance of the cells of interest.
Density gradient centrifugation, velocity sedimentation, and counterflow centrifugal elutriation are methods currently used to separate cells based on their physical properties such as size and density. While these methods work well as pre-enrichment steps, none are accurate and/or specific enough to yield pure populations of stem cells.
Flow cytometry is extremely sensitive because it looks at each cell individually. It can distinguish multiple markers, their relative level of expression, the size and granularity of each cell, and can sort out specific cells into a waiting tube. However, the equipment is highly sophisticated, the processing is relatively slow, and it is difficult to sterilize the instrument between samples.
By immobilizing the antibody on a solid phase, several methods have been used to process larger cell numbers in a relatively short time while still exploiting the specificity of the antigen/antibody interaction. Panning is an effective three-step technique for cell selection. First, mononuclear cells are separated from bone marrow, peripheral, or cord blood by density gradient centrifugation, negatively panned for soybean agglutinin binding, and positively panned with an anti-CD34 antibody-coated flask. The selected cells are released by mechanical agitation. The major problems with this method are the multiplicity of steps, subsequent low yields and the surface area required to give clinically useful stem cell numbers.
Another method along these lines uses magnetic beads as a solid support. Mononuclear cells are incubated with anti-CD34 antibody and bound to magnetic beads coated with sheep anti-mouse antibodies. The CD34+ cells are pulled to the side of the tube by applying a magnet. Historically, it has been difficult to dislodge the beads from the cells without harming the cells and perhaps of greater concern is that incomplete removal of the beads prior to reinfusion may be harmful to the patient.
One clinical technique is cell separation by avidin-biotin column chromatography as developed by Berenson et al. White blood cells incubated with a biotinylated anti-CD34 antibody are applied to a column containing avidin-coated polyacrylamide or agarose beads. After washing, the bound CD34+ cells are stripped from the bed by mechanical agitation. Theoretically, the mechanical release breaks the link at its weakest point, the antigen/antibody, and leaves the CD34 antigen intact on the cell surface. This is proposed because of the high affinity between biotin and avidin. Cells selected from bone marrow, peripheral blood, and cord blood by this method have been allogenically (unrelated donor) or autologously (self) transplanted into more than 200 patients worldwide to treat breast cancer, neuroblastoma, non-Hodgkin's lymphoma, and chronic myelogenous leukemia.
There are a number of innate problems facing any rare cell separation system. For instance, large surfaces tend to bind cells nonspecifically, thus decreasing specificity. Whereas low specificity is easy to overcome in systems where large numbers of the desired cells are available, it becomes a critical factor in separating stem cells since they are in such low abundance and in a heterogeneous cell background. Detachment is also a problematic stage in separation procedures. Detachment methods currently employed include the use of enzymes that chemically clip the cells from the solid phase, mechanical methods which tear them off, and polyclonal antibodies which compete them off.
A highly purified population of stem cells is necessary for a variety of in vitro experiments and in vivo indications. For instance, a purified population of stem cells will allow for identification of growth factors associated with their self-regeneration. In addition, there may be as yet undiscovered growth factors associated (1) with the early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the negative control of stem cell proliferation.
A comparison between current cell selection methods has proven that the use of antibodies to target specific cell populations consistently results in higher specificity and recovery when compared to non-antibody based methods. However, the current methods cannot produce pure cell populations (purity≦70%) and cannot recover more than 65% of the target cells (de Wynter et al., 1995). All current methods require the sample to be collected, often frozen and transferred to the laboratory for cell selection/depletion. None of the existing devices has the capacity to perform “in line” cell selection, that is, to directly select cells from normal peripheral blood as it is being drawn from a donor. Such possibility would make stem cell donation as simple as blood donation and would have enormous implications in the field of stem cell transplantation. It would tremendously increase the number of potential donors and expand the histocompatibility repertoire of stem cells available for transplantation, probably making the concept of stem cell banks a reality. The subject invention provides an efficient system for stem cell purification.